Enzyme coagulants such as rennin and pepsin are used extensively as milk coagulants in cheesemaking.
Rennin is formed from the inactive precursor, prorennin. The process, which has not been investigated in detail, is quite rapid below a pH of 3.0. Whether enzymatic activation by rennin itself occurs has not been established with certainty, although there is some indication that the process is autocatalytic. The molecular weight of rennin is about 40,000, and the enzyme has been obtained in crystalline form. The isoelectric point is about 4.5, which is considerably higher than that of pepsin. The pH optimum for activity of the enzyme with hemoglobin is 3.7 and its maximum stability in solution is near a pH of 5.5 to 5.8.
The enzyme, which has many similarities to pepsin is present in the lining of the fourth stomach of the calf. It is important to distinguish between the enzyme rennin and the extracts which are produced from the stomachs of calves, young lambs, or young goats. Rennin is the proteolytic agent of milk clotting found in rennet, but other enzymes may be present in the crude commercial extracts. If the enzyme is obtained from the stomach of calves which have been fed anything but milk, it will be contaminated with pepsin.
Although rennet is the preferred enzyme, other animal enzymes present in animal organs, such as stomachs, can be extracted and are useful for coagulating. For example, the enzyme pepsin comprises about 10% of the active coagulant found in veal organs, about 65% of the active coagulant in bovine organs and up to 100% of the active coagulant found in hog organs. Thus, it can be appreciated, the commercial extracts of animal coagulants will vary in the amount of enzymes present at a standard level of coagulating activity. Thus, the term enzyme coagulants is used in the description of the present invention.
Listed in Table I below are typical assays of various animal coagulants and their usage rates relative to standardized rennet which was assigned a usage factor of 1.00. Usage factors of other animal coagulants are based on the ratio of 12% reconstituted powdered milk/whole milk of the sample to the 12% reconstituted powdered milk/whole milk of the rennet standard. The % of Active Rennet (A.R.) describes the relative activity of each sample compared to that of the rennet standard in whole milk at a pH of 6.5. Thus, the theoretical 12% reconstituted powdered milk/whole milk coagulating units (RU) for a sample can be established to obtain equivalent activity in the cheese vat to rennet.
TABLE I __________________________________________________________________________ Reconstituted Dry Milk(12%) Whole Milk Reconstituted % of A.R. at pH 6.3 at pH 65. Dry Milk/ Usage Equivalent Sample RU/ml RU/ml Whole Milk Factor Activity __________________________________________________________________________ Standard Veal 100 31.6 3.16 1.00 100 Standard Bovine 242 30.6 7.91 2.67 97 Standard Bovine 236 35.0 6.75 2.15 111 Fungal - Type A 168 32.4 5.18 1.65 99 Fungal - Type B 124 33.5 3.70 1.16 104 50% Veal/50% Bovine 260 63.2 4.10 1.34 192 50% Veal/50% Bovine 310 71.2 4.35 1.40 219 __________________________________________________________________________
When rennet is obtained from the fourth stomach of young calves, either fresh or dried strips of the mucosa cut from the stomach are used. One method of preparing rennin in powder form is to mince the mucosa, adjust to pH 2.0 to 3.0 with hydrochloric acid, and incubate the thin slurry at 42.degree. C. (108.degree. F.) to convert the zymogen, prorennin, to rennin. The pH is next adjusted to 5.5 with sodium phosphate, and the whole fluid mass dried in a vacuum and powdered. Fat may be removed from the dry powder by solvent extraction.
Another method of preparing rennin comprises stirring for several days at pH 5.2, the dried and ground, or the minced tissue with 5% to 10% sodium chloride solution containing 2% to 4% of boric acid or other preservative. The extract is strained from the undissolved tissue, acidified with hydrochloric acid to ph 2.5, and allowed to stand for about 3 days. This activates the prorennin and coagulates mucin and other colloidal matter. The extract, after adjustment to pH 5.3 to 6.3, wherein rennin is most stable, is clarified by filtration. It may be evaporated at low temperature under vacuum and is marketed as a single, double, or triple strength rennet extract. A powder is obtained by precipitating with excess sodium chloride or sodium sulfate at the isoelectric point, pH 3.6, and allowing it to stand for 12 hours, filtering and drying. It is sold as standardized rennet powder.
Liquid rennet preparations generally have a pH between 5.6 and 5.8 in order to provide the most stable environment. The rennet extract can be sterilized by filtration through filters which retain bacteria, and other methods of sterilization by acids, iodine, or by heating have been suggested. Rennet contains considerable amount of pepsin if stomachs of older calves or cows are included with the stomachs of suckling calves. If the calves are fed anything else but milk, the stomach extracts will also contain pepsin. In many countries the stomachs of sheep and goats (or lambs and kids) are used. Extraction of such stomachs can be carried out with 10% acetic acid solutions by treating 100 g of the stomach with 500 ml of the acid five times at 30.degree. C. and during a 24 hour period. The extracts are combined, filtered, and vacuum concentrated to 20 ml.
The commercial extraction of rennet heretofore included washing and drying the rennet for 3 to 6 days and grinding the rennet with cellulosic fiber, such as excelsior, wood chips, for up to a day and one-half, adding water and countercurrent extracting for 2 to 6 days. These steps are repeated until the standardized strength is obtained. The residue had little utility due to the presence of wood chips, etc. Some methods today use reverse osmosis to concentrate the extract followed by an ultrafiltration step.
Most commercial processes presently on stream subject the various enzyme coagulants to hostile conditions relative to stability. For example, various air drying procedures used to dewater animal organs, prolonged counter current extractions, although somewhat effective in achieving release of these enzyme coagulant from the animal organs, create hostile conditions for these sensitive enzyme coagulants. The result is that reductions in yield are attributed in part to loss of enzyme coagulants due to instability.
It has been found that air drying of rennets results in loss of activity as shown in Table II below.
TABLE II ______________________________________ AIR DRIED - vs - VACUUM DRIED - vs - UNDRIED RENNET FOR EXTRACTION Treatment % Activity % Recovery ______________________________________ Rennets Dried 11/2 days in air drier. Ground in hammer mill and extracted overnight at pH 6.0 38.5 87.5 Same lot of rennets dried in vacuum drier for 24 hours. Ground and extracted as above. 45.5 100.00 Same lot of rennets-ground in meat grinder to a fine grind. Extracted as above. 45.5 100.00 ______________________________________
It has been observed that certain pre-treatments of the animal organs prior to extraction have no noticeable effect on activity. For example, grinding the organs to various mesh sizes does not appear to affect the activity level, provided the organs are fresh or frozen and not allowed to stand at room temperature for prolonged periods. This is shown in Table III below.
TABLE III ______________________________________ % Activity Type of Animal Organ Type of Grinder Used at 16 Hrs. ______________________________________ Bovine Stomach (Source A) Meat Grinder fitted 58 with 1/8" head Bovine Stomach (Source A) Meat Grinder fitted 60 with 1/16" head Bovine Stomach (Source B) Meat Grinder fitted 38 with 1/8" head Bovine Stomach (Source B) Meat Grinder fitted 40 with 1/16" head ______________________________________